Journal: Reproductive biology and endocrinology : RB&E

Article Title: Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

doi: 10.1186/s12958-021-00880-4

Figure Lengend Snippet: Fig. 3 Expression analysis of SLO3 mRNA and protein and localization of the SLO3 protein in sperm flagella. A Quantitative real-time PCR analysis of the expression of SLO3 mRNA in the spermatozoa from normal control and the SLO3-mutated individual. Compared with control, the expression of SLO3 mRNA is significantly reduced in the sperm from the SLO3-mutated individual. β-actin was used as an internal control. Data are presented as the mean ± SEM. ***P < 0.001; Student’s t-test. B Immunoblotting of sperm lysates from a normal control and the SLO3-mutated individual using an anti-SLO3 antibody. Na+/K+-ATPase α1 was used as loading control. C Representative images of spermatozoa from controls and from the SLO3-mutated individual stained with anti-SLO3 antibody, anti-Ac-tubulin antibody, and Hoechst. SLO3 staining is concentrated at the mid-piece of sperm flagella and faintly along the flagella in the fertile control but is significantly reduced in the sperm flagella of the individual harbouring the SLO3 mutation. Scale bar, 20 μm

Article Snippet: Membranes were then immunoblotted using the following primary antibodies: rabbit polyclonal anti-LRRC52 (1:1000; PA5–107159, Invitrogen, Carlsbad, CA 92008 USA), rabbit polyclonal anti-CatSper1 (1:1000; DF9349, Affinity Biosciences, Beijing, China), rabbit polyclonal anti-Na+/K+-ATPaseα1 (1:2000; ABP51894, Abbkine, China), anti-HSP60 antibody (1:1000; ab13532, Abcam, Cambridge, UK), and HRP-conjugated β-actin (1:2000; HRP-60008, Proteintech, Rosemont, IL, USA), at 4 °C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining, Mutagenesis

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

doi: 10.1186/s12958-021-00880-4

Figure Lengend Snippet: Fig. 5 Effects of SLO3 deficiency on mitochondrial function. A Influence of the SLO3 mutation on mitochondrial membrane potential (MMP). Assessment of mitochondrial membrane potential by flow cytometry acquisition for JC-1-stained (a marker of MMP) cells was performed through FL1 for green and FL2 for red fluorescence. MMP is significantly reduced in mutant sperm compared with control. B Immunoblotting of sperm lysates from controls and the SLO3-mutated individual using the anti-HSP60 antibody. β-actin was used as loading control. C The expression of HSP60 is almost diminished in SLO3 mutant spermatozoa. HSP60 staining is present in the flagellar midpiece of spermatozoa from normal controls, but significantly reduced in spermatozoa from A-132. Scale bars, 20 μm

Article Snippet: Membranes were then immunoblotted using the following primary antibodies: rabbit polyclonal anti-LRRC52 (1:1000; PA5–107159, Invitrogen, Carlsbad, CA 92008 USA), rabbit polyclonal anti-CatSper1 (1:1000; DF9349, Affinity Biosciences, Beijing, China), rabbit polyclonal anti-Na+/K+-ATPaseα1 (1:2000; ABP51894, Abbkine, China), anti-HSP60 antibody (1:1000; ab13532, Abcam, Cambridge, UK), and HRP-conjugated β-actin (1:2000; HRP-60008, Proteintech, Rosemont, IL, USA), at 4 °C overnight.

Techniques: Mutagenesis, Membrane, Flow Cytometry, Staining, Marker, Fluorescence, Control, Western Blot, Expressing

Journal: Theranostics

Article Title: Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

doi: 10.7150/thno.69885

Figure Lengend Snippet: Figure 1. RBP-J decoy oligodeoxynucleotides (ODNs) inhibited Notch signaling activation. (A) The structure of the decoy ODNs for the transcription factor RBP-J. (B–D) RAW264.7 cells or HUVECs were transfected with RBP-J decoy ODNs (Decoy RBP-J) or control decoy ODNs (Decoy Ctrl). After 24 h, Notch signaling was activated by the overexpression of the Notch intracellular domain (NICD) (Ad-NICD) in RAW264.7 cells or with Dll4-Fc in HUVEC cells. The mRNA levels of Notch target genes were detected by qRT-PCR in RAW264.7 cells (B) or HUVECs (C). The expression of Hes1 in RAW264.7 cells was determined by western blot, with β-actin serving as a reference control (D). (E) Luciferase reporter assay. HeLa cells were co-transfected with the RBP-J reporter plasmid pGa9816, pEFBOS-NICD, and increasing concentrations of RBP-J decoy ODNs; pRL-TK served as a reference control. Luciferase activity was detected 24 h after transfection. (F) Representative electrophoretic mobility shift assay (EMSA) results showing the binding of RBP-J to RBP-J decoy ODNs. HeLa cells were transfected with pCMV-RBP-J-Flag. Next, cell extracts were incubated in EMSA/Gel Shift binding buffer in the presence of biotin-labeled RBP-J decoy ODNs, unlabeled RBP-J decoy ODNs, control decoy ODNs, or an anti-Flag antibody, as indicated. Bars = means ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The samples were analyzed by sodium dodecyl sulfate (SDS)–PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated first with primary antibodies targeting Hes1, CD9, Alix, flotillin-1 (Cell Signaling Technology), HEY1, β-actin (Proteintech, Wuhan, China), and VDAC1 (Abcam, Cambridge, UK) and then with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Zhuangzhi Bio, Xi’an, China).

Techniques: Activation Assay, Transfection, Control, Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Gel Shift, Labeling

Journal: Theranostics

Article Title: Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

doi: 10.7150/thno.69885

Figure Lengend Snippet: Figure 4. Exosomes loaded with RBP-J decoy oligodeoxynucleotides (ODNs) inhibited Notch signaling in macrophages. Bone marrow-derived macrophages (BMDMs) were incubated with Dil-exosomes (A) or exosomes loaded with FAM-labeled ODNs (B) for 24 h and then analyzed by fluorescence microscopy. Nuclei were counterstained with Hoechst 33258. (C) Exosomes were loaded with oligonucleotides (miR-140-5p mimics or control mimics, 100 pmol) by electroporation and different concentrations of the loaded exosomes were incubated with BMDMs. After 24 h, the level of miR-140-5p was quantified in the BMDMs by qRT-PCR, with U6 serving as a reference control. (D–F) BMDMs were incubated with exosomes that had been electroporated with RBP-J decoy or control decoy ODNs for 24 h and then stimulated with LPS (200 ng/mL) for 24 h. The mRNA levels of Hes1, IL1β, IL6, and iNOS in BMDMs were detected by qRT-PCR (D). The protein level of Hes1 was determined by western blot (E), with β-actin serving as a reference control. The levels of IL1β and IL6 in culture supernatant were detected by ELISA (F). (G, H) Mice were intraperitoneally injected with CCl4 twice a week for 6 weeks; exosomes loaded with RBP-J decoy or control decoy ODNs (exosomes/decoy ODNs = 200 ng/2.5 nmol) were infused into mice four times via tail vein injection. F4/80+ hepatic macrophages were isolated using magnetic-activated cell sorting (MACS). The protein level of Hes1 was determined by western blot (G) and the mRNA levels of Hes1, Cyld, IL1β, and IL6 in F4/80+ hepatic macrophages were measured by qRT-PCR (H). Bars = means ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The samples were analyzed by sodium dodecyl sulfate (SDS)–PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated first with primary antibodies targeting Hes1, CD9, Alix, flotillin-1 (Cell Signaling Technology), HEY1, β-actin (Proteintech, Wuhan, China), and VDAC1 (Abcam, Cambridge, UK) and then with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Zhuangzhi Bio, Xi’an, China).

Techniques: Derivative Assay, Incubation, Labeling, Fluorescence, Microscopy, Control, Electroporation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Isolation, FACS